Means and method of producing and controlling cultures of microorganisms



Sept. 4, 1956 A. Gor-:TZ 2,761,813

MEANS AND METHOD OF' PRODUCING AND CONTROLLING CULTURES 0F' MICROORGANISMS Filed Jan. 21, 1955 2 Sheets-Sheet 1 By nu M Arran/vins Sept. 4, 1956 A. GoETz 2,761,813

MEANS AND METHOD OF PRODUCING AND CONTROLLING CULTURES OF' MICROORGANISMS Filed Jan. 21, 1953 2 sheets-snm 2 SELECTED MxcooRoAmsms AVAILABLE DYE j STIM ULATING NUTRIENT MICRO Ummm? UNAVAILABLE DYE w M m A r\ f\ f\ A f\ r\ n r\ A -o-o-o-o-o-o-o- FIG. 7 FIG. 8 .F1629 sELEcTnvE "I: NuTRlEH-r l V33: roxxc Mm' RIAL D Q Q Z I E Z) (ANTIBxo-nc .2F/3ra!! INVENTOR.

AM [xA/V05@ G0572 BY w M United States Patent() MEANS AND METHOD OFA PRDUCINGI AND CONTROLLING CULTURES F MICRO- ORGANSMSv Alexander Goetz, Altadena, Calif.

Application January 21, 1953, Serial No. 332,283

18 Claims. (Cl. 19S-80) My invention relates to means and methodA of producing and controlling cultures of microorganisms and the ob- 'ects of my invention are:

First, to provide a means and method'- of producing cultures of microorganisms which is applicable to all types of microbiological investigations,l whether simple, routine, or complex and extensive, and whether concerning bacteria, yeasts, fungi, or other microorganisms capable of growth.

Second, to provide a means and method of this classv which virtually eliminates the excessive amount' of laboratory equipment, particularly glassware, and the attendant problems of sterilization of such equipment.

Third, to provide a means and method of this class wherein the laboratory labor is drastically reduced and simpliiied so that relatively unskilled' persons may conduct even involved investigations concerning microorganisms in far less time and sterile equipment with more dependable results than have been possible heretofore;

Fourth, to provide a means and method of this class which is particularly suited for investigations of microorganisms made necessary by disaster or Warfare, in' that the necessary information can be obtained Without a laboratory and at an earlier time, to permit eiiectivev counter measures.

Fifth, to provide a means and method ofv this class wherein special kits and nutrient media may be prefabricated by carefully controlled methods of mass production, then sterilized and stored Vfor months or years until needed, such kits containing all the components necessaryr to con'-V duct a particular investigation or test.

Sixth, to provide a means and' method ofthis-type which utilizes a microporous membrane capable of co'l'- lecting microorganisms on its surface, and special nutrient pads or membranesplaced under the microlilter'inwetting contact to feed the microorganisms.

Seventh, to provide a means a-nd method of' producing cultures of microorganisms whereby the nutrients, in-A hibitors, antibiotics, or other substance intendedv to grow, selectively inhibit or kill the microorganisms maybe fed; automatically in a predetermined sequence and in precisely the desired amounts so that the results obtained`y may be depended upon to be accurate and complete.

With the above and other objects in view as may appear hereinafter, reference is directed to the' accompanying drawings in which:

Figure l is a sectional view showing an assembly or packet incorporating my invention.

Figure 2 is an exaggerated and diagrammatic sectional' view` showing one of the units for producing biologica-ll cultures.

Figure 3- is a sectional view of one of the nutrient units illustrating the manner in which the nutrient pad fs sup-V plied with the quantity of water required for uniform re hydration.

Figure 4 is a fragmentary' plan View of one of the filter cups employed in my invention.

Figure 5 is a bottom view of onev of the nutrient cups;-v

Patented Sept. 4, 1956 rice:

Figure 6 is a fragmentary sectional view ofv a filter cup andi associated apparatus employed to draw a liquid sampleH through the filter cup.

Figures 7', 8 and 9 are fragmentary diagrammatic sectional view of a iil'ter membrane and nutrient pad in which the nutrient pad is laminated and its component laminae provided with different growthk control media.

Figure 7 illustrates the initial conditionsV existing at the time the filter membrane and nutrient pad are brought into Contact.

Figure 8"Y illustrates an intermediate condition.

Figure 9 illustrates a nal condition.

Figure l0 is another fragmentary diagrammaticv sectional view illustrating another type oi'laminated nutrient pad'.

FigureA l1 is a plan view of another' of the laminae of the nutrientrpadillustrating the manner in: which several growth' control media or test substancesmay be applied to laminae components of the pad.

Figure l2v is: a diagrammatical view showing atypical arrangementv facilitating early detection of microorganisms or the effect of inhibitors thereon.

Reference is firstV directed to Figures l-5, which illustrate devices employed to perform my methodof producing bacteriological cultures.

In order to conduct a-bacteriol'ogical investigation a set of' sterile filter cups 1 is' employed. Each filter cup' may be formed of paper or other inexpensive expendable material. Each filter cup` has a tapered sidewall and a flat bottornwhich iscu-tf out, leaving a marginal flange. Cement-'edi and sealed: by its periphery to the under side of the ilter cup isa microtilter membrane or molecular filter membrane 2.

A filter membrane found suitable for thepurposes of my invention consists of al homogenous. screen-like rigid cellulose orcellulose ester lm devoid of any hbrous struc:-l ture and of a highly uniform porosity (80 to 90% ),.and having almost complete wet-dry stability. Such membrane now available has a large number (flg/cm) of fine pores which permit quantitative surface retention: of bacterial forms at 'ovv rates of i003 m1./'c`m.2'/ While the exact structure of the filter membrance cannot bev determined with accuracy, it has been established that itv is' anisotropic; that is, liquids move preferentially normal to the" surfaces of the membrane with only minorl lateral' diifusion. Also, the pore diameters arey smallest at the v upper surface and the upper surface is quite smoothso that the bacteria or other microorganisms retained do not penetrate into the membrane but lie on the surface. For the purposes of this invention the lter membrane per se may beconsidered asV conventional; however, its combi nation with the other elements of'my invention does' con-- sti'tute'" a part of my present invention.

As a companion to the lter cup I there is` provided' a sterile nutrientV cup 3, also formed of paper or similar' expendablev material. The nutrient cup has divergingf side Walls; the bottom is hat and perforated as indicated by 4. Cemented by its' marginal portion tothe perforated bottom of the nutrient cup is a nutrient pad` 5, which isv formed of absorbent material, such as a heavy blotting paper.

The nutrient pad, before or after attachment to the nutrient cup, -is impregnated With a'nutrient solution of poses hereinafter described, should have V no selective ad slorptive quality. For some purposes the nutrient pad may be' isotropic; i. e., the Water or other liquid diffuses or migrates'in all directions equally. For other purposes :the

liquid normal to the surfaces of the pad wlth minimal lateral diffusion. Thus, for some purposes, a microlter membrane or a modication thereof may be used.

The term nutrient, as herein used, is intended t designate any substance which may be fed to the microorganism; thus, it may be a growth-promoting or a partiaily growth-inhibiting substance. A solution of nutrient is absorbed by the pad and the pad is then dehydrated and sterilized under precautions avoiding denaturation of the nutrient. The quantity as well as the quality of the nutrient can be held to close tolerances.

Many nutrients are conventionally employed in bacteriological studies or studies of other microorganisms. They may be simple or complex mixtures designed for specific purposes, and often contain selective inhibiting agents for certain types of microorganisms; thus,\the conventional nutrient formulas number in the hundreds or thousands. For the purposes of the present invention the nutrients per se, or more broadly the growth controlling substances per se, whether nutritive or inhibitory in nature, maybe considered as conventional. However, as an element or elements of my combination the nutrient pad, or various arrangements of said nutrient pad, and therconcept of an automatic nutrient schedule, as hereinafter set forth, are part of my present invention.

The nutrient cup and filter cup are so proportioned that the filter cup sets in the nutrient cup with its microfilter membrane in uniform contact with the nutrient pad as shown in Figure 2. The nutrient cups may be so arranged that they will nestle partially into the filter cups so as to form an air space 6 of predetermined volume. This may be accomplished by providing rolled edges 7 and 8 around the rims of the filter cups and nutrient cups, respectively, to form shoulders or stops as shown in Figure 1.

Prior to use the nutrient pad is rehydrated with the desired quantity of water merely by oating the nutrient cup on the surface of a quantity of sterile water as suggested in Figure 5.

'I'he perforations in the bottom of the nutrient cups are of such number and size that the water will be transferred uniformly over the area of the nutrient pad without appreciable transverse flow within the boundaries of the pad so that the concentration of the nutrient will remainl uniform throughout the pad. p

Since the capillary volume capacity of the pad islimited, the pad will take up a defined quantity of water; thus, there is no need to measure the water volume required for rehydration and for producing the optimal nutrient concentration.

A sterile cup 9 similar to the nutrient or lter Cups, that is, made of paper of similar expendable material, maybe employed as a container for the distilled water required for rehydration.

It should be observed that the manufacture of the microlter membrane can be so controlled, that upon wetting the membrane, it can be caused to either shrink or expand slightly. For the purposes of my invention it is ,desirable that the microlter membrane shrink slightly on being wetted so that when brought in contact with the nutrient pad it will not buckle or warp, but instead be inuniform wetting contact over its entire area.

'During storage the lter cups and nutrient cups may bestacked alternately as shown in Figure l. The bottom of the lowermost nutrient cup is preferably protected by a rigid base member 10 having a marginal ange. The

, water cup 9 may be the uppermost cup contained in the stack. The water cup, being of the same dimensions as the nutrient cup, will form the desired uniform air space .6 above the Vuppermost filter cup. A cap or cover 11 having a marginal flangerts over the water cup. The

entire stack of cups is sealed in a bag 12.

The bag 12 may be formed of polyethylene or another plastic iilm impermeable to moisture but permeable to ethylene oxide or formaldehyde, to permit sterilization :after assembly. The reinforcing base member 10 and ycover 11 protect the kit against crushing. Initially the kit comprising the stack of lter cups and nutrient cups, together with the water cup, is sealed in the bag 12 and sterilized.

In conducting an investigation of microorganisms the iilter cups are seated, one at a time, in 'a iilter funnel 13, 'a's shown in Figure 6. The funnel is provided with a shoulder 14.V Seated in the shoulder is a porous supporting disk 15 which may be in the form of a porous plate of fritted coke, glass, metal or the like; that is, any porous material which provides adequate support for the microfilter membrane. The stem of the funnel 13 may extend through a stopper 16 into a flask 17, which is adapted to be connected to a source of vacuum pressure. It Ishould be noted that the construction of funnel and filter cup provides for a tight seal at 14 so that Ia vacuum in the flask will'cause the fluid in the cup to pass quantitatively through the membrane. The filter cup may serve as a means of measuringthe quantity of liquid to be passed through the filter membrane. For this purpose graduations 18 may be printed on the wall of the lter cup.

The microorganisms Iare retained on the top surface of the microlter. It is of course desirable that the number of microorganisms collected be within an optimum range in order that a dependable count may be made. Thus the quantity of water or other liquid to be tested is regulated accordingly, or if the microorganisms are excessive, the liquid is suitably diluted with sterile water.

The nutrient pad is wetted las shown in Figure 3 and the corresponding microilter membrane is placed on top as shown in Figure 2. As pointed out before, care is taken to provide wetting contact between the lower surface of the microiilter membrane and the nutrient pad throughout their areas.

It will be observed that the arrangement of the various elements of my apparatus is such that at any time during the operation the sterility of the equipment by handling is assured, i. e., it is unnecessary to take speci-al precautions for aseptic handling.

After the bacteria have been collected on the membrane surface in the filter cup, and the former has been brought in wetting contact with the rehydrated nutrients in .the

nutrient cup, the pairs of cups are restacked and have an air space between the surface of the membrane and the bottom of the next nutrient cup, suicient for theg'rowth of the microorganisms.

After the microorganisms have been collected on the series of microlter membranes and theseA membranes have been restacked on their respective nutrient pads, the entire kit may be returned to the bag 12 and then subjected to incubation at the desired temperature. It Will be observed that all of the factors necessary for growth of the microorganisms may be controlled with a high` one component in order to provide automatically a timed sequence or schedule of more than one nutrient formula to the microorganisms on the membrane surface. Purpose and utility of a scheduled feeding of nutrients may be illustrated by the following example:

In sanitary water tests it is customary to culture bacteria contained in a water sample by. the addition of nutrients which inhibit the development of the majority Qf the microfibra except vthosewhich are originating from sewage. This group, known as E. coli, is related also' to certain other bacterial groups such as B. aerogenes; These groups lall can be nutriated by lactose and are least sensi# tive to certain dyes such as fuchsine, brilliant green, etc. Nutrient pads designed for the selective nutrition of the coli-aerogenes group would thus contain in suitable concentration lactose in addition to other components as nutrient, and a dye, e. g., fuchsine, as the inhibiting factor for the development of the rest of the bacterial ora in the water sample.

It is also known that the application of this nutrientdye combination to such bacteria when deposited by ltration on a microlilter membrane can produce an insutlicient or even no growth development on the mem; brane, apparently because the inhibitory dye aiects the less viable bacteria, though of the coli-aerogenes type, adversely during the early stages of development on the membrane. The nal count may thus be misleadingly small or entirely fail to indicate the presence of these bacteria.

This disadvantage can be avoided by contacting the membrane for the first two or three hours of its growth development during incubation with a nutrient pad which does not contain vthe dye but rather a nutrient formula of highly stimulating composition, able to bring even weakened bacterial ceils into the logarithmic growth phase wherein the viability is high and the tolerance for the diiferentiating dye is substantially increased. The membrane, after this stage is reached, is then transferred to another nutrient pad which contains the nutrientY and the dye, which latter inhibits further growth on all but the' aforementioned bacteria. It has to be realized tha-t the size of bacterial colonies increases exponentially withtime so that the size of the colonies of` those bacteria which are inhibited by the transfer to the second nutrient pad has, after two or three hours, not reached into anA order of magnitude visible by subsequent counting methods. Errors in counting due to the presence of even a large number of subsequently inhibited colonies do not occur if the transfer to the second nutrient pad is not effected too late.

This method has, especially for large scale routine operation, the disadvantage of requiring an additional manipulation of the membrane due to the transfer from' one nutrient pad to another. This transfer involves ex-v posure of the culture and thus possible additional contamination, and may cause severe growth retardation by partial drying, if not expertly handled.

This difliculty may be avoided by converting the operation of transfer into an automatic nutrient schedule effected by the use of a laminated nutrient pad consisting in' this case of two separate component leaves as sche# matically represented in Figures 7-9. The upper pad 20 contains for this -spec'iiic application to the coli-aerogenes group' a generally stimulating lactose (or dextrose)-'pep tone nutrient; the lower pad 21 of larger liquid capacity c'fo'ntains a' lactose-pep'tone nutrient with fuchsine as inhibitor. Both leaves are separately impregnated and subsequently dehydrated, and finally attached to each other to form one unit.

Furthermore, the material constituting the upper pad has the property of sorbing the dye up to a certain quantity without releasing it subsequently, in other words, the upper pad has the capacity for eliminating the dimolecules from the solution yielded by the lower pad and rendering the dye temporarily unavailable to the membrane 2. Figure 7 indicates the initial situation prior to hydration, where the membrane 2 carries on its surface the retained bacterial cells from the preceding iiltration 1of the sainple, the' upper pad 2b the components of the stimulating nutrient (-H, the lower pad 21 the lactose nutrient andthe inhibitory dye (o).

Upon contact with sterile water by the lower side of the pad'271 as describedV above, and upon contact between 2,

20' and 21 the results schematically 'shown in Figure 8 take' place: the water taken up b'y 21 brings its components (-o') into solution. This initial solution, due to capillary action, reaches gradually into the upper laminate 20 where it dissolves in addition the nutrient components contained therein. However, due to the sorptive qualities of 20, the dye component (o) originating from 21 is removed from the solution and rendered unavailable, as indicated by (o), as it appears at the interface between 2 and 20. The membrane 2, having smaller capillaries than the nutrient pad components, removes the top layer of this solution from 20 and lifts it to its upper surface where the solution reaches the bacteria. They are thus contacted with a highly stimulating nutrient in the absence of the dye and begin to grow.

As the microorganisms metabolize gradually, the so conditioned nutrient has to diffuse upward in order to maintain an equilibrium in the assembly. As a consequence, the components supplied by 20 will gradually become exhausted and so will be the adsorptive capacity for the dye. The latter will pass now in an available state from 21 through 20 into 2 and a gradual change of nutrient composition will affect the growth of bacteria. The initial general stimulation will be replaced by selective growth inhibition and growth nutrition of lactose fermenters capable of resisting the dye. This final stage of nutrition, reached in the nal phases of incubation, is schematically shown in Figure 9.

The time at which this state will be reached will depend upon: (a) the relative liquid capacity of the materials forming the leaves 20 and 2i; (b) the diusion rate through these materials; (c) the initial concentration of solutes in Ztl and 21 respectively; (d) the sorptive capacity for the dye applied and, if any,- of the membrane material. All these variables can be controlled by the thickness and type of material of the nutrient pad laminate components and the concentration of nutrients initially applied prior to dehydration.

The use of such preimpregnated dehydrated sterile nutrient pads will effect several economies: the operating laboratory does not have to prepare nutrients; the opera'tor does not have to manipulate liquid nutrients in the field, thus avoiding the ever present danger of accidental contamination and misleading results; also a timed nutrient schedule, if prefabricated, is accurately reproducible, provided that the main factor which controls the diffusion and adsorption rate, that is to say, the incubation temperature, is closely maintained. The main saving, however, consists in the practically complete avoidance of the requirement for sterile glassware.

In the above specific example, the upper laminate component acted in a twofold manner, for it added stimulating nutrients to the solution from the other pad and it subtracted certainv inhibitory components temporarily. i do not wish to restrict my invention to a combination of simultaneousl additive and subtractive performances. In order to illustrate the utility of a strictly additive performance of the upper laminate, I refer to the following example:

In certain clinical, diagnostic 'test performances, one aims at the earliest possible identification of one or more of a whole group of selectively antibiotic agents (sulfanilamides, pencillin, aureomycin, etc.) which will inhibit n the growth of the llora derived from the patient, in order to be able to resort to the proper chemotherapy even be` fore the detailed identification of the pathogenic organism has been possible. For this purpose one can impregnate all or part of the upper laminate of the nutrient pad with one or several solutions of diierent antibiotics in such a manner that the additional agents are restricted to certain fractional areas' of the top laminate. In this case it may be desirable to use, as a materialfor such a laminate, a leaf characteristized by a high diifusion rate normal and a low diffusion rate parallel to the surface of the leaf.

Y When the nutrient in the lower leaf is rehydrated and passes through the upper laminate, it dissolves, in analogy to the former example, the (antibiotic) components and carries to the microorganism a thus modified nutrient. Consequently a bacterial growth will occur on the upper surface of the membrane only in those regions where an antibiotic had been added to the nutrient which did not affect the Vparticular microorganisms in question. Correlating the various types of antibiotics with the fractional areas in the upper laminate which carry diterent antibiotics can very simply be effected by a system of markings on the filter membrane which detine the relative position of the lter membrane and the nutrient pad'laminate during incubation.

It will be noted that in the above example-contrary to the previous one-the selective inhibitor (antibiotic) was addedfto the nutrient locally during the initial nutritional phase, while in the first example, the selective inhibitor was added in a delayed manner, that is, at a later stage in the growth phase.

i wish to include in my invention another principle resulting in a delaying action especially when lan addition is required towards the end of the growth phase, that is, after sixteen to twenty-four hours. The utility of eecting such a delay is manifold. It may be desirable to stop further growth development after a given time of incubation, in which case a general bacterial poison (phenol, mercurous or silver compounds) should be released7 inhibiting any further growth whatsoever on the membrane. lt may also he desirable for the purpose of identification to release certain of such growth inhibitory dyes which act as pH or redox indicators and permit lthe differentiation of the bacterial colonies on the membrane by characteristic color reactions while the initial presence of such indicators inthe concentrations required would be partially or totally growth inhibiting.

This result can be accomplished in Vthe following manner: the agen-t is dissolved in a solution of an organocollo-id such as gelatin, agar, carboxymethylcellulose, and one laminate is impregnated with this solution and subsequently dehydrated. The agent (provided that a suitable concentration ratio between the organocolloidalsolute and the agent existed) will be encapsulated into the colloid after dehydration. Upon rehydration, a substantial period of swelling, etc., will 'be necessary before the agent becomes available for diusion into the surface of the iilter. By the proper choice `of the colloid and the aforementioned concentration ratio, a delaying action reaching over days if necessary, can be accomplished. Another application of the same delaying principle consists in `casting a iilm of the combination of the organosol and the agent which is subsequently dehydrated similar to the gelatin films used in the forma-tion of photographic emulsions. After drying, this iilm may then be used as a rthird laminate component.

I wish also to state as part of my invention that a sequence of impregnations of a nutrient pad leaf first with the 'organosol containing the agent, subsequent dehydration of same and subsequent impregnation of the same nutrient pad with an `aqueous solution of nutrients without retarding colloids followed by immediate dehydration of the second impregnation can produce similarly an automatic delaying action, in which case the nutrient solution will be immediately available upon rehydration, whereas -the delaying agent begins `to condition the nutrient solution much later, in spite of the coexistence of lthe two agents in the same nutrient pad.

Still further, the colloid containing the selected agent may be formed in a moisture-permeable iii-m and placed at the top or bottom of the nutrient pad.

In the examples given, two laminations are indicated; however, if more elaborate feeding schedules are desired, for purposes of particular investigations, three or more laminations might be employed. By way of example, reference is directed to Figure l0. In many investgations of microorganisms it is not only desirable to isolate as completely as possible a particular species of microorganisms but to determine what poison is most eiective against the microorganism in question. 1Eor example, it may be desired 'to determine which antibiotic is most effective against a particular bacterial species, even though the species is not identified.

By adding a third lamination 22 such a test may 'be made automatically. The third lamination may be impregnated or printed with an antibiotic, thus the nutrient schedule initially follows the course indicated -in Figures 7, 8 and 9 and terminates with 4the upward diiusion 'of the antibiotic, represented by check marks, to the surface of the microiilter.

It has been found, by control of the thickness and,

porosity of the laminations of the nutrient pad as well as the concentrations of the nutrients, inhibitors or the like, that any desired predetermined nutrient schedule can 'be provided. It thus follows that a reliable survey can be conducted by a person having no technical knowledge as long as simple instructions are followed.

So far in considering the nutrient pad, whether a single number or laminated, it has been considered as isotropic, that is, permitting movement of liquid therein either laterally `or parallel to its surfaces and vertically or normal to its surfaces. For many purposes it is desirable to utilize an anisotropic nutrient pad or an anistropic lamination of such pad; that is, to confine the movement of liquid therethrough to flow normal to its surfaces with no appreciable lateral diiusion. It will be noted that this is a characteristic of the microlilter itself.

If such a pad or lamination is used, it is possible to subject different areas of the microfilter to different agents and, in effect, enable one microlter membrane to serve :the place of several membranes, with the very great advantage of maintaining conditions exactly equal except for the precise variable that is to be investigated.

For example, if in Figure l0 the laminations were anisotropic, the lowermost lamination might be imprinted with several antibiotics as represented by the circular areas 23 in Figure ll. After completion of incubation of the microorganisms collected on the microiilter, it will be readily apparent which of the selected antibiotics is most effective. While an antibiotic test is selected as an example, it is of course understood that any other selected series of test substances may be similarly used.

It of course is not necessary that the multiple imprinted or treated lamination be the bottom one. Any or all the others may be similarly prepared. in many cases it is preferred to so prepare the upper lamination. Again selecting antibiotics as an example, the uppermost lamination may be imprinted or treated as indicated in Figure 1l. The nutrient common to all areas of the antibiotic treated lamination is conditioned differently in the diterent areas, making possible an early determination of which antibiotic to use.

The importance of early determination cannot be overemphasized for the reason that microorganisms develop in number logarithmically. Thus in the case of a patient with an infectious disease, the earlier the proper countermeasure can be determined the better. Without knowing the identity of the microorganism, its susceptibility to a large number of antibiotics may be determined in a remarkably short time, by microscopic examination of the microiilter membrane before the microorganisms have been fully incubated.

It should be observed that if a delayed supply of a particular series of agents is desired, the multiple imprinted lamination may be employed in conjunction with the dilerent methods of use previously described; that is, the selective sorpton, or delayed solution techniques may be used.

While the foregoing examples have dealt principally with bacteria, it should be observed that my means and method are not limited to the development of bac- Q teriologi'cal cultures only but are equally adapted to the study of fungi, molds, virus, bacteriophag'es and any her microorganisms capable of retention on the microter.

In this regard it should be observed that the smaller virus and bacteriophages are sufficiently small to pass through even the microilter. They may be entrapped, however, by first passing through -a filter a solution containing larger bacteria or other microorganisms which serve as a host to the virus or bacteriophage in question. A thin lter cake of such bacteria is coliected on the membrane, then the solution containing the virus or bacteriophage is drawn through. It is found that the virus or bacteriophage will be retained by the bacteria filter cake. A nutrient schedule is applied in the manner analogous to that hereinbefore described. Detection and analysis of the virus or bacteriophage is made by bservation of the voids produced in the bacteria filter cake.

It will be observed that the upward movement of the nutrient substance, as describedrhereinbefore, is a chromatographic action. However, the timing of the nutrient schedule may be controlled by use of substances which dissolve or return to solution at different rates; for example, water soluble carboxymethylcelluloses of different concentrations return to solution at different rates.

Again referring to the technique, wherein antibiotics or analogous substances are incorporated in the lamination immediately adjacent the microfilter, or, for that matter in the microfilter itself, a procedure whereby early detection may be made involves means indicated diagrammatically in Figure 12.

First, it should be observed that initially at least the microorganism growth is essentially two-dimensional, i. e., along the surface of the microfilter. Now, if the surface of the microlter membrane 24 is dyed black, preferably nonuorescent, the very early stages of growth (or lack of growth) may be determined by directing a low angle beam of light 25 across the surface of the microlter. The colonies of microorganisms become quite distinct as light scattering dots which may be observed by eye or by a microscope 26.

Not only does the technique enable detection at an extremely early stage, but it also permits repeated or continuous examination of the microorganisms during their development, including photographing without interfering with their development.

Having fully described my invention, it is to be understood that I do not wish to be limited to the details herein set forth, but my invention is of the full scope of the appended claims.

I claim:

1. A microorganism culture producing means, cornprising: a iilter cup having a microiilter membrane forming the bottom thereof; a nutrient cup having a nutrient impregnated disk of absorbent material forming the bottom thereof; said lter cup adapted to nestle in the nutrient cup with its undersurface in wetting contact with the upper surface of said nutrient pad.

2. A microorganism culture producing means, comprising: a filter cup having a microlter membrane forming the bottom thereof; a nutrient cup having a nutrient impregnated disk of absorbent material forming the bottom thereof; said cups having tapered side walls and adapted to be stacked alternately, said cups being so proportioned that each lter cup is adapted to nestle within a corresponding nutrient cup with its filter membrane in wetting contact with the surface of said nutrient pad; said nutrient cups adapted to nestle only partially into said filter cups whereby said nutrient pads are spaced from the lter pad below to form an atmospheric region thereabove of predetermined Volume.

3. A microorganism culture producing means, comprising: a porous microlilter membrane having an upper surface adapted to retain microorganisms; at least a pair of absorbent. members, said members being impregnated with dineren: soluble substances adapted to anser the g'iwth of microog'anism's; said members adapted to be wetted and placed in wetting contact with each othe and the side ofk said 4mi'croilter membrane opposite from its microorganism retaining surface, and cause the migration Of YSi'd Sl'lbS'ns lil S''q'nCe T hIllgh Said lCrllleI' said microorganisms.

4. A -iicroci-ga-iiisrill culture producing means, comprising: 'a porous microlilter membrane having an upper surface adapted to etai microorganisms; a lamination 'of 'absorbent members, each of said members being' impregnated Vwith a dilferent microorganism growth con'- tolliig substance soluble in water; said lamination of absorbent members adapted to be wetted and placed in wetting Contact with the side of said microiltei' opposite from said microorganisms, whereby said substances are supplied in sequence to said microorganisms.

5. A microorganism culture producing means, cbmpiisigza porous mirilter membrane having an upper 'sui-'fase adapted 'to fetain microorganisms; a lamination of absorbent members, each of said members being pregtd with a different microorganism growth con'i trolling substance soluble in water; said lamination of absorbent members adapted to be wetted and placed in wetting contact with the side of said microltr opposite from said microorganisms, whereby said substances are supplied in sequence to said microorganisms; one of said members being impregnated with a substance toxic to a selected class of microorganisms thereby todetermine the resistance of the selected microorganisms to said toxic i substance.

6. A microorganism culture producing means, comprising: a porous microlter membrane having an upper surface adapted to retain microorganisms; a lamination of absorbent members, each of said members being impregi'- nated with a different microorganism growth controlling substance soluble in water; said lamination of absorbent members adapted to be wetted and placed in wetting contact with the side of said microlter opposite said microorganisms, whereby said substances are supplied in sequence to said microorganisms; at least one of said ab sorbent members being impregnated in different regions with different growth controlling substances and said microlter membrane and lamination of absorbent members being anisotropic whereby microorganisms in corresponding regions of said microlter membrane are differently affected.

7. A microorganism culture producing means as set forth in claim 4, wherein: said microlilter membrane forms the bottom of a first cup member and said absorbent members are disposed in the bottom of a second cup member, the cup members being so proportioned that on nesting the rst cup member into the second cup member said microtilter membrane and absorbent members are brought into wetting contact; a plurality of said cup members adapted to be stacked alternately whereby the second cup members nest partially into said rst cup members to establish an atmospheric region of predetermined volume.

8. A microorganism culture producing means as set forth in claim 6, wherein: said microlter membrane forms the bottom of a rst cup member and said absorbent members are disposed in the bottom of a second cup member, the cup members being so proportioned that on nesting the rst cup member into the second cup member said microlter membrane and absorbent members are brought into wetting contact; a plurality of said cup members adapted to be stacked alternately whereby the second cup members nest partially into said lirst cup members to establish an atmospheric region of predetermined volume.

9. A microorganism culture producing means, comprising: an anisotropic microilter membrane adapted to collect microorganisms on a surface thereof; and a 1annnated pad including a lamination containing an initially dehydrated nutrient and an anisotropic lamination containing a plurality of substances in separate regionsthereof, said substances being selected for various eiects on microorganism growth, said pad adapted to be wetted and placed in wetting contact with said microlter to cause anisotropic migration of said substances to corresponding areas of said microlter.

l0. A microorganism culture producing means for supplying soluble substances to microorganisms collected on the surface of an anisotropic microtilter, comprising: a laminated pad of porous material, at least one of said laminations being anisotropic and carrying a plurality of soluble substances calculated to have diierent effects on microorganisms; another of said laminations containing a nutrient substance; said pad adapted When wetted and placed in wetting contact with said microlter to supply said substances to said microorganisms in accordance with a predetermined time schedule.

ll. A microorganism culture producing means for supplying soluble substances to microorganisms collected on the surface of an anisotropic microlter, comprising: a laminated pad, the various laminations containing diftferent dehydrated substances calculated to effect the growth of microorganisms, said pad adapted when wetted and placed in wetting contact with said microllter to supply said substances to rsaid microorganisms in timed sequence.

l2. A microorganism culture producing means, com prising: a microlter having a dark lightabsorbing upper surface adapted to collect microorganisms; a pad impregnated with dehydrated soluble substances calculated to arect said microorganisms, and adapted to be supplied thereto through said microiilterv when said pad is wetted and placed in wetting contact with the underside of said microiilter; and means for detecting said microorganisms as they 4'develop on the surface, including a light source directed at low angles across the upper surface of said microlter.

i3. A method ofv producing cultures of microorganisms, characterized by: collecting microorganisms on one surface of a filter membrane; and supplying in predetermined timed sequence through said lter membrane '12 from the opposite side thereof a series of soluble substances calculated to aiect the ydevelopment of cultures of said microorganisms.

14. A method of producing cultures of microorganisms, characterized by: collecting microorganisms on one surface of a iilter membrane; and supplying through different areas of said filter membrane from the opposite side thereof simultaneously a plurality of soluble substances calculated to aiect the development of cultures of said microorganisms present in corresponding areasof said filter membrane.

l5. A microorganism culture producing means as set forth in claim 4, wherein: said lamination of absorbent Y members comprises two laminae, the lamina adjacent said microtilter being impregnated with a generally stimulating nutrient tending to promote growth of substantially all microorganisms retained on the microilter, and said second lamina being impregnated with a substance tending relatively to inhibit certain classes of said micro' organisms.

16. A microorganism culture producing means as set forth in claim 4, wherein: said lamination of absorbent members comprises at least two laminae, the lamina adjacent said microlter being impregnated at least in part with a substance toxic to certain classes of said microorganisms, and the second lamina containing a nutrient.

l7. A microorganism culture producing means as set forth in claim 4, wherein: at least one of said absorbent members is impregnated with a growth controlling substance encapsulated into a colloid whereby upon wetting of said absorbent memberl said substance is retained by said colloid for a predetermined time before being supplied to said microorganisms.

i8. A microorganism culture producing means as set orth in claim 4, wherein: a moisture permeable colloidal film encapsulating a selected growth controlling substance is included in said lamination.

References Cited in the le of this patent FOREIGN PATENTS 957,354 VFrance Feb. 17, 1950 

1. A MICROORGANISM CULTURE PRODUCING MEANS, COMPRISING: A FILTER CUP HAVING A MICROFILTER MEMBER FORMING THE BOTTOM THEREOF; A NUTRIENT CUP HAVING A NUTRIENT IMPREGNATED DISK OF ABSORBENT MATERIAL FORMING THE BOT- 